U.S. patent number 6,743,599 [Application Number 10/106,665] was granted by the patent office on 2004-06-01 for compositions and assays utilizing adp or phosphate for detecting protein modulators.
This patent grant is currently assigned to Cytokinetics, Inc.. Invention is credited to Jeffrey T. Finer, Fady Malik, Roman Sakowicz, Christopher Shumate, Kenneth Wood.
United States Patent |
6,743,599 |
Finer , et al. |
June 1, 2004 |
**Please see images for:
( Certificate of Correction ) ** |
Compositions and assays utilizing ADP or phosphate for detecting
protein modulators
Abstract
Described herein are methods which identify candidate agents as
binding to a protein or as a modulator of the binding
characteristics or biological activity of a protein. Generally, the
methods involve the use of ADP or phosphate. The assays can be used
in a high throughput system to obviate the cumbersome steps of
using gels or radioactive materials.
Inventors: |
Finer; Jeffrey T. (Foster City,
CA), Malik; Fady (Burlingame, CA), Sakowicz; Roman
(Foster City, CA), Shumate; Christopher (San Francisco,
CA), Wood; Kenneth (Foster City, CA) |
Assignee: |
Cytokinetics, Inc. (South San
Francisco, CA)
|
Family
ID: |
32328827 |
Appl.
No.: |
10/106,665 |
Filed: |
March 25, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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724990 |
Nov 28, 2000 |
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314464 |
May 18, 1999 |
6410254 |
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Current U.S.
Class: |
435/21; 435/15;
435/16; 435/17; 435/18; 435/19; 435/196; 435/23; 435/24 |
Current CPC
Class: |
C12Q
1/42 (20130101); C12Q 1/48 (20130101); C12Q
1/50 (20130101) |
Current International
Class: |
C12Q
1/48 (20060101); C12Q 1/50 (20060101); C12Q
1/42 (20060101); C12Q 001/42 (); C12Q 001/48 ();
C12Q 001/52 (); C12Q 001/50 (); C12N 009/18 () |
Field of
Search: |
;435/21,15-19,23-24,196 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Field et al., "A novel genetic system to detect protein-protein
interactions," Nature, 340:245-246 (1989). .
Vasavada et al., "A contingent replication assay for the detection
of protein-protein interactions in animal cells," PNAS USA,
88:10686-10690 (1991). .
Fearon et al., "Karyoplasmic interaction selection strategy: A
general strategy to detect protein-protein interactions in
mammalian cells," PNAS USA, 89:7958-7962 (1992). .
Dang et al., "Intracellular Lecucine Zipper Interactions Suggest
c-Myc Hetero-Oligomerization" Mol. Cell. Bio., 11:954-962 (1991).
.
Chien et al., "The Two-hybrid system: A method to identify and
clone genes for proteins that interact with a protein of interest,"
PNAS USA, 88: 9578-9582 (1991). .
Webb, A continous spectrophotometric assay for inorganic phosphate
and for measuring phosphate release kinetics in biological systems,
PNAS USA, 89-4884-4887 (1992). .
Ungerer et al., "An enzymatic assay of inorganic phosphate in serum
using nucleoside phosphorylase and zanthine oxidase," Elsevier
Clinica Chimic Act., 223: 149-157 (1993). .
Rieger et al., "A continuous spectrophotomeric assay for asparte
transcarbamylase and ATPases," Anal., Biochem., 246:86-95 (1997).
.
Brune et al., :Direct, real-time measurement of rapid inorganic
phosphate release using a novel fluorescent probe and its
application to actomyosin subfragment 1 ATPase, Bio chem.,
33:8262-8271 (1994). .
Hackney, "The rate-limiting step in mocrotubule-stimulated ATP
Hydrolysis by Dimeric Kinesin Head Domains Occurs while bound tot
he microtubule," J. Biol. Che., 269(23):16508-16511 (1994). .
Greengard, "Determination of intermediary metabolites by enzymic
fluorimetry," Nature, 178:632-634 (1956). .
Banik et al., "A continous fluorimetric assay for ATPase activity,"
Biochem. J., 266:611-614 (1990). .
Fiske et al., "The colorimetric determination of phosphates,"
J.Bio. Chem., 66:375-400 (1925). .
Goldstein, "With apologies to Scheherazade: Tails of 1001 Kinesin
Motors," Annu. Reb. Genet., 27:319-351 (1993). .
Desai et al., "Kin I Kinesins are microtubule-destablizing
Enzymes," Cell, 96:69-78 (1999). .
Walczak et al., "XKCM1: A zenopus kinesin-related protein that
regulates microtubule dynamics during mitotic spindle assembly,"
Cell, 84:37-47 (1996). .
Ronaghi et al., "Real-time DNA Sequencing Using Detection of
Pyrophosphate Release," Analytical Biochemistry, 242: 8-89 (1996).
.
Wood et al., "Plus End-Directed Microtubule Motor Required for
Chromosome Congression," PCT application claiming priority to
U.S.S.N 60/058,645 filed Sep. 11, 1997. .
Ting-Guang Hunag and David D. Hackney, Drosophila Kinesin Minimal
Motor Domain Expressed in Escherichia coli, The Journal of
Biological Chimistry, Jun. 10, 1994, pp. 16493-16501, vol. 269, No.
23, U.S.A. .
Stedman's Medical-Dictionary, p. 1404, 26th Edition, Williams &
Wilkins, A Waverly Company..
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Primary Examiner: Saidha; Tekchand
Attorney, Agent or Firm: Stevens; Lauren L. Townsend and
Townsend and Crew LLP
Parent Case Text
CROSS-REFERENCES TO RELATED APPLICATIONS
This application is a continuation of U.S. application Ser. No.
09/724,990, filed Nov. 28, 2000, abandoned, which is a continuation
of U.S. application Ser. No. 09/314,464, filed May 18, 1999, now
U.S. Pat. No. 6,410,254, the disclosure of which is incorporated
herein by reference.
Claims
What is claimed is:
1. A method for rapidly identifying a compound that modulates
polymerization or depolymerization of a cytoskeletal filament
protein, wherein said filament protein comprises actin or tubulin,
said method comprising: a) adding a compound to a mixture
comprising said filament protein under conditions which normally
promote either polymerization or depolymerization of said filament
protein; b) adding a motor protein to said mixture, wherein said
motor protein has ATPase activity, and said ATPase activity is
activated by polymerized filament protein, and said motor protein
is added to said mixture under conditions which normally allow the
production of ADP or phosphate by said motor protein; and c)
determining the ATPase activity of said motor protein by detecting
the formation of ADP or phosphate, wherein a change in ATPase
activity in the presence of said compound compared to the absence
of said compound is an indication that said compound is a modulator
of filament protein polymerization or depolymerization.
2. The method of claim 1, wherein said determining occurs by a
fluorescent, luminescent, radioactive, or absorbance readout.
3. The method of claim 1, wherein said activity of said motor
protein is determined at multiple time points.
4. The method of claim 1, wherein a plurality of compounds are
added.
5. The method of claim 4, wherein a plurality of compounds and a
plurality of motor proteins are added.
6. The method of claim 1, wherein said compounds are added using a
robotic system.
7. The method of claim 1, wherein the motor protein is a
kinesin.
8. The method of claim 7, wherein the motor protein is Kin2,
chromokinesin, Kif1A, KSP, CENP-E, MCAK, HSET, or Kif15.
9. The method of claim 1, wherein the motor protein is myosin.
10. The method of claim 1, wherein the step of determining the
activity of said motor protein comprises coupling an enzymatic
reaction that utilizes ADP or phosphate to the oxidation of NADH;
and measuring NADH oxidation as a measure of ADP production.
11. The method of claim 1, wherein an increase in the activity of
said motor protein in the presence of said compound compared to the
absence of said compound is an indication that said compound
promotes polymerization of said filament protein, and a decrease in
the activity of said motor protein in the presence of said compound
compared to the absence of said compound is an indication that said
compound promotes depolymerization of said filament protein.
Description
FIELD OF INVENTION
This invention is related to the use of adenosine diphosphate (ADP)
or phosphate in assays for identifing compounds which bind to or
modulate the binding characteristics or biological activity of a
protein.
BACKGROUND OF THE INVENTION
Drugs and other compounds intended for use in the diagnosis, cure,
mitigation, treatment or prevention of disease in man or other
animal or for use in the agricultural arena, have made a
significant impact on the practice of modem medicine and on the
agricultural arena. In some cases, such as in the development of
vaccines, drugs have essentially eradicated once untreatable
diseases. In the case of the agriculture, compounds have been
developed which both extend the life and/or volume of produce as
well as kill unwanted plants where desirable. Therefore, the
development of these compounds is of great interest.
Many useful compounds modulate the physical interaction of
proteins. Traditionally, these protein-protein interactions have
been evaluated using biochemical techniques, including chemical
cross-linking, co-immunoprecipitation, co-fractionation and
co-purification. Recently genetic systems have been invented to
detect protein-protein interactions. The first work was done in
yeast systems, and was termed the "yeast two-hybrid" system. The
basic system requires a protein-protein interaction in order to
turn on transcription of a reporter gene. Subsequent work was done
in mammalian cells. See Fields et al., Nature 340:245 (1989);
Vasavada et al., PNAS USA 88:10686 (1991); Fearon et al., PNAS USA
89:7958 (1992); Dang et al., Mol. Cell. Biol. 11:954 (1991); Chien
et al., PNAS USA 88:9578 (1991); and U.S. Pat. Nos. 5,283,173,
5,667,973, 5,468,614, 5,525,490, and 5,637,463.
In another approach to drug discovery, studies are designed to
determine the biological activity of a protein. For example, the
conditions such as the specific substrate or stimulator required
for an enzymatic reaction are investigated. Moreover, there are a
number of studies designed specifically for aide in the detection
step in these assays. For example, one study discloses a
spectrophotometric assay for inorganic phosphate (Pi) to probe the
kinetics of Pi release from biological systems such as GTPases and
ATPases. Webb, PNAS, 89:4884-4887 (1992). Another study reports on
an enzymatic assay of inorganic phosphate in serum using nucleoside
phosphorylase and xanthine oxidase. Ungerer, et al., Elsevier
Clinica Chimica Act, 223:149-157 (1993). A continuous
spectrophotometric assay for aspartate transcarbamylase and ATPases
is reported on in Rieger, et al., Anal. Biochem., 246:86-95 (1997).
There is also a study which reports on the measurement of inorganic
phosphate release using fluorescent probes and its application to
actomysin subfragment 1 ATPase. Brune, et al., Biochem.,
33:8262-8271 (1994). U.S. Pat. No. 4,923,796 discloses a method for
quantitative enzymatic determination of ADP. Microtubule-stimulated
adenosine triphosphate (ATP) hydrolysis by kinesin is discussed in
Hackney, J. Biol. Chem., 269(23):16508-16511 (1994). Furthermore,
enzymatic fluorimetry and fluorimetric assays for ATPase activity
are reported on in Greengard, Nature, 178:632-634 (1956) and Utpal
and Siddhrtha, Biochem. J., 266:611-614 (1990), respectively.
In a different approach, modulators of an enzymatic reaction are
investigated, wherein the conditions which allow the enzymatic
reaction to occur are already known. For example, U.S. Pat. No.
5,759,795 discloses an assay for identifying an inhibitor of a
Hepatitis C Virus NS3 protein ATPase which involves a luciferase
reaction. Luciferase reactions are known in the art. In the case of
an ATPase inhibitor, the presence of an ATPase inhibitor is
indicated when ATP is available to drive the oxidation of luciferon
by luciferase. This approach requires ATP but does not re-generate
ATP.
Thus, while efforts have been made toward drug discovery, more
efficient means are desirable. In particular, there is a need for
an efficient system which can distinguish between a compound
directly binding to a second component, or whether the compound
modulates the binding between two other components, or whether the
compound modulates the biological activity of a known enzymatic
reaction. Accordingly, it is an object of the present invention to
provide methods of identifying compounds which either bind to or
which modulate the binding characteristics or the biological
activity of a target protein. It is also an object to provide
compositions for use in the assays provided herein.
SUMMARY OF THE INVENTION
The present invention provides methods which identify candidate
agents that bind to a a protein or act as a modulator of the
binding characteristics or biological activity of a protein. In one
embodiment, the method is performed in plurality simultaneously.
For example, the method can be performed at the same time on
multiple assay mixtures in a multi-well screening plate as further
described below. Furthermore, in a preferred embodiment,
fluorescence or absorbance readouts are utilized to determine
enzymatic activity. Thus, in one aspect, the invention provides a
high throughput screening system.
In one embodiment, the present invention provides a method of
identifying a candidate agent as a modulator of the activity of a
target protein. The method comprises adding a candidate agent to a
mixture comprising a target protein which directly or indirectly
produces ADP or phosphate under conditions which normally allow the
production of ADP or phosphate. The method further comprises
subjecting the mixture to an enzymatic reaction which uses said ADP
or phosphate as a substrate under conditions which normally allow
the ADP or phosphate to be utilized and determining the level of
activity of the enzymatic reaction. A change in the level between
the presence and absence of the candidate agent indicates a
modulator of the target protein.
In one aspect, the target protein indirectly produces the ADP or
phosphate by producing a substrate for a reaction which produces
the ADP or phosphate. In another aspect, the target protein
indirectly produces phosphate or ADP or phosphate by regulating an
enzyme which produces ADP or phosphate. In yet a further aspect,
the target protein directly produces phosphate or ADP.
In another aspect, the invention provides a method of identifying a
candidate agent as a modulator of the activity of a target protein
wherein the target protein uses ADP or phosphate directly or
indirectly. The method comprises adding a candidate agent to a
mixture comprising the target protein under conditions which
normally allow the utilization of ADP or phosphate. The method
further comprises determining the level of utilization wherein a
change in the level between the presence and absence of the
candidate agent indicates a modulator of the target protein.
In another embodiment provided herein is a method for identifying
whether any two target proteins interact. The method comprises
providing a first target chimera comprising a functional molecular
motor binding domain and a first target protein. The method further
comprises providing a second target chimera comprising a functional
microtubule stimulated ATPase domain and a second target protein.
Additionally, the method comprises combining the first and second
target chimeras under conditions which normally allow activity of a
motor protein which comprises a molecular motor binding domain and
a microtubule stimulated ATPase domain, wherein an increase in
motor protein activity indicates interaction between the two target
proteins.
In a further embodiment a method is provided for identifying
whether a candidate agent is a modulator of at least one of any two
target proteins. The method comprises providing a first target
chimera comprising a functional molecular motor binding domain and
a first target protein and further providing a second target
chimera comprising a functional microtubule stimulated ATPase
domain and a second target protein. Additionally, the method
comprises combining the first and second target chimeras in the
presence and absence of a candidate, wherein a change in motor
protein activity, which requires both a molecular motor binding
domain and a microtubule stimulated ATPase, between the presence
and absence of a candidate agent indicates the candidate agent is a
modulator of at least one of the target proteins.
Additionally, provided herein is a chimeric protein comprising a
functional molecular motor binding domain and a target binding
domain wherein the chimeric protein is independent of a functional
microtubule stimulated ATPase domain. Also provided herein is a
chimeric protein comprising a functional microtubule stimulated
ATPase domain and a target binding domain, wherein the chimeric
protein is independent of a functional molecular motor binding
domain.
In one aspect, a nucleic acid comprising a nucleic acid encoding a
chimeric protein in accordance with the present invention is
provided. In another aspect a cell comprising a nucleic acid or a
chimeric protein in accordance with the present invention is
provided.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention provides methods for the identification of
candidate agents that bind to a target protein or serve as
modulators of the biological activity of a target protein. These
assays utilize various methods to measure, in ways amenable to high
throughput screening, the generation or consumption of ADP or
phosphate. That is, target proteins that either directly or
indirectly produce or consume ADP or phosphate may be screened in
the present invention. Thus, by providing assay systems that
rapidly, efficiently and inexpensively assay ADP or phosphate,
modulators (including both antagonists and agonists) of any test
protein that directly or indirectly produces ADP or phosphate may
be found. The present invention thus utilizes high throughput
assays that obviate the traditional cumbersome steps of using gels
or radioactive materials.
Accordingly, the present invention provides methods of screening of
target proteins. By "target protein" herein is meant a protein that
directly or indirectly produces ADP or phosphate. The target
proteins can be from eukaryotes or procaryotes, including mammals,
fungi, bacteria, insects, and plants, as well as viruses. In a
preferred embodiment, the target proteins are from mammalian cells,
with rodents (mice, rats, hamsters, guinea pigs and gerbils being
preferred), primates and humans being preferred, and humans being
particularly preferred.
"Protein" in this context means a compound that comprises at least
two covalently attached amino acids and includes proteins,
polypeptides, oligopeptides and peptides. The proteins may be made
up of naturally occurring amino acids and peptide bonds, or
synthetic peptidomimetic structures. Thus "amino acid", or,
"peptide residue", as used herein means both naturally occurring
and synthetic amino acids. For example, homo-phenylalanine,
citrulline and noreleucine are considered amino acids for the
purposes of the invention. "Amino acid" also includes imino acid
residues such as proline and hydroxyproline. The side chains may be
in either the (R) or the (S) configuration. In the preferred
embodiment, the amino acids are in the (S) or L-configuration. If
non-naturally occurring side chains are used, non-amino acid
substituents may be used, for example to prevent or retard in vivo
degradations.
Suitable target proteins, include, but are not limited to,
cytoskeletal proteins including, but not limited to, kinesins,
myosins, tubulins, actins, tropomyosins, and troponins, with human
proteins being preferred.
In a preferred embodiment, the target protein is a kinesin,
including mitotic kinesins. Mitotic kinesins are enzymes essential
for assembly and function of the mitotic spindle, but are not
generally part of other microtubule structures, such as nerve
processes. Mitotic kinesins play essential roles during all phases
of mitosis. These enzymes are "molecular motors" that translate
energy released by hydrolysis of ATP into mechanical force which
drives the directional movement of cellular cargoes along
microtubules. The catalytic domain sufficient for this task is a
compact structure of approximately 340 amino acids. During mitosis,
kinesins organize microtubules into the bipolar structure that is
the mitotic spindle. Kinesins mediate movement of chromosomes along
spindle microtubules, as well as structural changes in the mitotic
spindle associated with specific phases of mitosis. Experimental
perturbation of mitotic kinesin function causes malformation or
dysfunction of the mitotic spindle, frequently resulting in cell
cycle arrest. From both the biological and enzymatic perspectives,
these enzymes are attractive targets for the discovery and
development of novel anti-mitotic chemotherapeutics.
Suitable kinesins include, but are not limited to, Kin2,
chromokinesin, Kif1A, KSP, CENP-E, MCAK, HSET and Kif15 is
provided. K335, Q475, D679, FL1, P166, H195, FL2, E433, R494, E658,
L360, K491, S553, M329, T340, S405, V465, T488, M1, M2, M3, M4, M5,
M6, FL3, A2N370, A2M511, K519, E152.2, Q151.2, Q353, M472 and
MKLP1. It is understood that unless a particular species is named,
the term "kinesin" includes homologs thereof which may have
different nomenclature among species. For example, the human
homolog of Kif1A is termed ATSV, the human homologue of Xenopus Eg5
is termed KSP, and human HSET corresponds to Chinese hamster
CHO2.
By "kinesin protein activity" or grammatical equivalents herein is
meant one of kinesin protein's biological activities, including,
but not limited to, its ability to affect ATP hydrolyzation. Other
activities include microtubule binding, gliding,
polymerazation/depolymerazation (effects on microtubule dynamics),
binding to other proteins of the spindle, binding to proteins
involved in cell-cycle control, or serving as a substrate to other
enzymes, such as kinases or proteases and specific kinesin cellular
activities such as chromosome congregation, axonal transport,
etc.
Methods of performing motility assays are well known to those of
skill in the art (see, e.g., Hall, et al. (1996), Biophys. J., 71:
3467-3476, Turner et al., 1996, Anal. Biochem. 242 (1):20-5; Gittes
et al., 1996, Biophys. J. 70(1): 418-29; Shirakawa et al., 1995, J.
Exp. Biol. 198: 1809-15; Winkelmann et al., 1995, Biophys. J. 68:
2444-53; Winkelmann et. al., 1995, Biophys. J. 68: 72S, and the
like).
In a preferred embodiment, the target protein directly or
indirectly produces ADP and/or phosphate. Included in the
definition of adenosine diphosphate (ADP) are ADP analogs,
including, but not limited to, deoxyadenosine diphosphate (dADP)
and adenosine analogs. As used herein, phosphate is used
interchangeably with inorganic phosphate.
In a preferred embodiment, the target protein directly produces ADP
or phosphate. In a preferred embodiment, the target protein is an
enzyme having activity which produces ADP and/or phosphate as a
reaction product. For example, proteins which directly produce ADP
include but are not limited to ATPases, kinases, GTPases,
phosphatases and phosphorylases. Suitable ATPases include, but are
not limited to, myosins, kinesins, dyneins, DNA gyrase, DNA
helicase, topoisomerase I and II, Na+-K+ ATPase, Ca2+ ATPase, F1
subunit of ATP synthase, terminase/DNA packaging protein; recA,
heat shock proteins, NSF, katanin, SecA, 5-lipoxygenase, and actin.
Suitable kinases include, but are not limited to, tyrosine kinases;
serine-threonine kinases; receptor tyrosine kinases; growth factor
receptors including but not limited to insulin receptor, epidermal
growth factor receptor, platelet derived growth factor receptor and
fibroblast growth factor receptor; ErbB2; calmodulin dependent
protein kinases; protein kinase A; protein kinase C; myosin light
chain kinase; CDK2 kinase; ROCK1 kinases; Src kinases;
phosphorylase kinase; CheA; adenylate kinase; glycolytic kinases;
EIF-2 alpha protein kinases; and Abl. Suitable GTPases include, but
are not limited to, G proteins, Rho family GTPases: cdc42, RalA,
RhoA and Rac1; Ras proteins; elongation factors including
EF1.alpha., EF1.beta..gamma., EF-Tu and EF-G; septins; tubulin; ARF
related GTPase; rab; SSRP receptor; rhodopsin; transducin; and
GTPase activating protein (GAP). Suitable phosphatases include, but
are not limited to, protein phosphatases; myosin phosphatase; IP3
phosphatase; pyrophosphatase; and Cdc25. Suitable phosphorylases
include, but are not limited to, polynucleotide phosphorylase and
glycogen phosphorylase.
By "ATPase" herein is meant an enzyme that hydrolyzes ATP. For
example, ATPases include proteins comprising molecular motors such
as kinesins, myosins and dyneins. "Molecular motor" is a molecule
that utilizes chemical energy to produce mechanical force or
movement; molecular motors are particularly of interest in
cytoskeletal systems. For further review, see, Vale and Kreis,
1993, GUIDEBOOK TO THE CYTOSKELETAL AND MOTOR PROTEINS New York:
Oxford University Press; Goldstein, 1993, Ann. Rev. Genetics 27:
319-351; Mooseker and Cheney, 1995, Annu. Rev. Cell Biol. 11:
633-675; Burridge et al., 1996, Ann. Rev. Cell Dev. Biol. 12:
463-519.
In one embodiment, the target protein indirectly produces ADP or
phosphate. In one aspect, a target protein indirectly produces ADP
or phosphate by producing a product that then serves as a substrate
in a subsequent enzymatic reaction for producing ADP or phosphate.
For example, in a preferred embodiment, the target protein can be a
pyrophosphate producing enzyme. Suitable pyrophosphate producing
enzymes include, but are not limited to, DNA polymerases; RNA
polymerases; reverse transcriptase; DNA ligase; adenylate cyclase;
guanylate cyclase; PRPP synthetase; TRNA synthetases; acyl CoA
synthetase and acetyl CoA carboxylase. Similarly, some ATPases
produce AMP that can then be used to make ADP.
In another embodiment, the target protein is a synthase. Thus,
preferred substrates for producing phosphate include pyrophosphate
and any of the mono-, di- and triphosphate versions of CTP, UTP,
GTP, ATP, and TTP, as well as derivatives including dideoxy
derivatives. Additionally, other sources of substrates that can be
cleaved to phosphate include phosphorylated peptides,
oligonucleotides, carbohydrates, lipids, etc. For example, inositol
triphosphate (IP3) is an important signaling moiety. Accordingly,
any target protein which produces these compounds or others that
can be used to produce phosphate or ADP may be assayed using the
methods of the present invention.
In another aspect, a target protein indirectly produces ADP or
phosphate by regulating an enzyme which produces phosphate or ADP.
For example, the target can be an activator of an ATPase, such as
an actin filament or a microtubule; thus in this embodiment, the
target protein may be a protein polymer or oligomer. Alternatively,
the target protein can be a filament binding protein or regulatory
protein. For example, the regulatory protein can be the
troponin-tropomyosin complex which regulates the binding of myosin
to actin. Since myosin's ATPase is activated by binding to actin,
modulators of this regulatory protein complex can be identified by
the methods provided herein.
In a preferred embodiment, the target protein may consume ADP or
phosphate; that is, rather than looking for an increase in signal,
a loss of signal may be monitored.
Also included within the definition of the target proteins of the
present invention are amino acid sequence variants of wild-type
target proteins. These variants fall into one or more of three
classes: substitutional, insertional or deletional variants. As for
the target proteins as discussed below, these variants ordinarily
are prepared by site specific mutagenesis of nucleotides in the DNA
encoding the target protein, using cassette or PCR mutagenesis or
other techniques well known in the art, to produce DNA encoding the
variant, and thereafter expressing the DNA in recombinant cell
culture as outlined above. However, variant target protein
fragments having up to about 100-150 residues may be prepared by in
vitro synthesis using established techniques. Amino acid sequence
variants are characterized by the predetermined nature of the
variation, a feature that sets them apart from naturally occurring
allelic or interspecies variation of the target protein amino acid
sequence. The variants typically exhibit the same qualitative
biological activity as the naturally occurring analogue, although
variants can also be selected which have modified characteristics
as will be more fully outlined below.
While the site or region for introducing an amino acid sequence
variation is predetermined, the mutation per se need not be
predetermined. For example, in order to optimize the performance of
a mutation at a given site, random mutagenesis may be conducted at
the target codon or region and the expressed variants screened for
the optimal combination of desired activity. Techniques for making
substitution mutations at predetermined sites in DNA having a known
sequence are well known, for example, M13 primer mutagenesis and
PCR mutagenesis. Screening of the mutants is done using assays of
target protein activities.
Amino acid substitutions are typically of single residues;
insertions usually will be on the order of from about 1 to 20 amino
acids, although considerably larger insertions may be tolerated.
Deletions range from about 1 to about 20 residues, although in some
cases deletions may be much larger.
Substitutions, deletions, insertions or any combination thereof may
be used to arrive at a final derivative. Generally these changes
are done on a few amino acids to minimize the alteration of the
molecule. However, larger changes may be tolerated in certain
circumstances. When small alterations in the characteristics of the
target protein are desired, substitutions are generally made in
accordance with the following chart:
CHART I Original Residue Exemplary Substitutions Ala Ser Arg Lys
Asn Gln, His Asp Glu Cys Ser Gln Asn Glu Asp Gly Pro His Asn, Gln
Ile Leu, Val Leu Ile, Val Lys Arg, Gln, Glu Met Leu, Ile Phe Met,
Leu, Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp, Phe Val Ile, Leu
Substantial changes in function or immunological identity are made
by selecting substitutions that are less conservative than those
shown in Chart I. For example, substitutions may be made which more
significantly affect: the structure of the polypeptide backbone in
the area of the alteration; for example the alpha-helical or
beta-sheet structure; the charge or hydrophobicity of the molecule
at the target site; or the bulk of the side chain. The
substitutions which in general are expected to produce the greatest
changes in the polypeptide's properties are those in which (a) a
hydrophilic residue, e.g. seryl or threonyl, is substituted for (or
by) a hydrophobic residue, e.g. leucyl, isoleucyl, phenylalanyl,
valyl or alanyl; (b) a cysteine or proline is substituted for (or
by) any other residue; (c) a residue having an electropositive side
chain, e.g. lysyl, arginyl, or histidyl, is substituted for (or by)
an electronegative residue, e.g. glutamyl or aspartyl; or (d) a
residue having a bulky side chain, e.g. phenylalanine, is
substituted for (or by) one not having a side chain, e.g.
glycine.
The variants typically exhibit the same qualitative biological
activity, although variants also are selected to modify the
characteristics of the target proteins as needed. Alternatively,
the variant may be designed such that the biological activity of
the target protein is altered. For example, glycosylation sites may
be altered or removed.
Further included within the definition of the target proteins of
the invention are covalent modifications of the target proteins.
One type of covalent modification includes reacting targeted amino
acid residues of a target protein with an organic derivatizing
agent that is capable of reacting with selected side chains or the
N- or C-terminal residues of a target protein. Derivatization with
bifunctional agents is useful, for instance, for crosslinking the
target protein to a water-insoluble support matrix or surface.
Commonly used crosslinking agents include, e.g., 1,1
-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,
N-hydroxy-succinimide esters, for example, esters with
4-azidosalicylic acid, homobifunctional imidoesters, including
disuccinimidyl esters such as
3,3'-dithiobis(succinimidylpropionate), bifunctional maleimides
such as bis-N-maleimido-1,8-octane and agents such as
methyl-3-[(p-azidophenyl)dithio]propioimidate.
Other modifications include deamidation of glutaminyl and
asparaginyl residues to the corresponding glutamyl and aspartyl
residues, respectively, hydroxylation of proline and lysine,
phosphorylation of hydroxyl groups of seryl or threonyl residues,
methylation of the .alpha.-amino groups of lysine, arginine, and
histidine side chains [T. E. Creighton, Proteins: Structure and
Molecular Properties, W. H. Freeman & Co., San Francisco, pp.
79-86 (1983)], acetylation of the N-terminal amine, and amidation
of any C-terminal carboxyl group.
Another type of covalent modification of the target proteins
included within the scope of this invention comprises altering the
native glycosylation pattern of the polypeptide. "Altering the
native glycosylation pattern" is intended for purposes herein to
mean deleting one or more carbohydrate moieties found in the target
native sequence, and/or adding one or more glycosylation sites that
are not present in the native sequence.
Addition of glycosylation sites to target polypeptides may be
accomplished by altering the amino acid sequence thereof. The
alteration may be made, for example, by the addition of, or
substitution by, one or more serine or threonine residues to the
native sequence (for O-linked glycosylation sites). The target
amino acid sequence may optionally be altered through changes at
the DNA level, particularly by mutating the DNA encoding the target
polypeptide at preselected bases such that codons are generated
that will translate into the desired amino acids.
Another means of increasing the number of carbohydrate moieties on
the target polypeptide is by chemical or enzymatic coupling of
glycosides to the polypeptide. Such methods are described in the
art, e.g., in WO 87/05330 published 11 Sep. 1987, and in Aplin and
Wriston, CRC Crit. Rev. Biochem., pp. 259-306 (1981).
Removal of carbohydrate moieties present on the targget polypeptide
may be accomplished chemically or enzymatically or by mutational
substitution of codons encoding for amino acid residues that serve
as targets for glycosylation. Chemical deglycosylation techniques
are known in the art and described, for instance, by Hakimuddin, et
al., Arch. Biochem. Biophys., 259:52 (1987) and by Edge et al.,
Anal. Biochem., 118:131 (1981). Enzymatic cleavage of carbohydrate
moieties on polypeptides can be achieved by the use of a variety of
endo- and exo-glycosidases as described by Thotakura et al., Meth.
Enzymol., 138:350 (1987).
Another type of covalent modification of target proteins comprises
linking the target polypeptide to one of a variety of
nonproteinaceous polymers, e.g., polyethylene glycol, polypropylene
glycol, or polyoxyalkylenes, in the manner set forth in U.S. Pat.
Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or
4,179,337.
Target polypeptides of the present invention may also be modified
in a way to form chimeric molecules comprising a target protein
fused to another, heterologous polypeptide or amino acid sequence,
a preferred embodiment of which is is described more fully below.
In one embodiment, such a chimeric molecule comprises a fusion of a
target polypeptide with a tag polypeptide which provides an epitope
to which an anti-tag antibody can selectively bind. The epitope tag
is generally placed at the amino- or carboxyl-terminus of the
target polypeptide. The presence of such epitope-tagged forms of a
target polypeptide can be detected using an antibody against the
tag polypeptide. Also, provision of the epitope tag enables the
target polypeptide to be readily purified by affinity purification
using an anti-tag antibody or another type of affinity matrix that
binds to the epitope tag.
Various tag polypeptides and their respective antibodies are well
known in the art. Examples include poly-histidine (poly-bis) or
poly-histidine-glycine (poly-his-gly) tags; the flu HA tag
polypeptide and its antibody 12CA5 [Field et al., Mol. Cell. Biol.,
8:2159-2165 (1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7
and 9E10 antibodies thereto [Evan et al., Molecular and Cellular
Biology, 5:3610-3616 (1985)]; and the Herpes Simplex virus
glycoprotein D (gD) tag and its antibody [Paborsky et al., Protein
Engineering, 3(6):547-553 (1990)]. Other tag polypeptides include
the Flag-peptide [Hopp et al., BioTechnology, 6:1204-1210 (1988)];
the KT3 epitope peptide [Martin et al., Science, 255:192-194
(1992)]; tubulin epitope peptide [Skinner et al., J. Biol. Chem.,
266:15163-15166 (1991)]; and the T7 gene 10 protein peptide tag
[Lutz-Freyermuth et al.; Proc. Natl. Acad. Sci. USA, 87:6393-6397
(1990)].
As will be appreciated by those in the art, the target proteins can
be made in a variety of ways, including both synthesis de novo and
by expressing a nucleic acid encoding the target protein.
Numerous suitable methods for recombinant protein expression,
including generation of expression vectors, generation of fusion
proteins, introducing expression vectors into host cells, protein
expression in host cells, and purification methods are known to
those in the art and are described, for example, in the following
textbooks: Sambrook et al., Molecular Cloning: A Laboratory Manual
(New York: Cold Spring Harbor Laboratory Press, 1989), Ausubel et
al., Short Protocols in Molecular Biology (John Wiley & Sons,
Inc., 1995), Harlow and Lane, Antibodies: A Laboratory Manual (New
York: Cold Spring Harbor Laboratory Press, 1988), O'Reilly et al.,
Baculovirus Expression Vectors: A Laboratory Manual (New York:
Oxford University Press, 1994), Richardson, Baculovirus Expression
Protocols (Totowa: Humana Press, 1995), Kriegler, Gene Transfer and
Expression: A Laboratory Manual (New York: Oxford University Press,
1991), Roth, Protein Expression in Animal Cells, Methods in Cell
Biology Vol. 43 (San Diego: Academic Press, 1994), Murray, Gene
Transfer and Expression Protocols, Methods in Molecular Biology,
Vol. 7 (Clifton: Humana Press, 1991), Deutscher, Guide to Protein
Purification, Methods in Enzymology Vol. 182 (San Diego: Academic
Press, Inc., 1990), Harris and Angal, Protein Purification Methods:
A Practical Approach (Oxford: IRL Press at Oxford University Press,
1994), Harris and Angal, Protein Purification Applications: A
Practical Approach (Oxford: IRL Press at Oxford University Press,
1990), Rees et al., Protein Engineering: A Practical Approach
(Oxford: IRL Press at Oxford University Press, 1992) and White, PCR
Protocols, Methods in Molecular Biology, Vol. 15 (Totowa, Humana
Press, 1993).
The selection of host cell types for the expression of target
proteins will depend on the target protein, with both eukaryotic
and procaryotic cells finding use in the invention. Appropriate
host cells include yeast, bacteria, archebacteria, fungi, plant,
insect and animal cells, including mammalian cells. Of particular
interest are Drosophila melangaster cells, Saccharomyces cerevisiae
and other yeasts, E. coli, Bacillus subtilis, SF9 cells (and other
related cells for use with baculoviral expression systems), C129
cells, 293 cells, Neurospora, BHK, CHO, COS, Dictyostelium,
etc.
In a preferred embodiment, the target proteins are purified for use
in the assays, as outlined herein, to provide substantially pure
samples. By "substantially pure" or "isolated" herein is meant that
the protein is unaccompanied by at least some of the material with
which it is normally associated in its natural state, preferably
constituting at least about 0.5%, more preferably at least about 5%
by weight of the total protein in a given sample. A substantially
pure protein comprises at least about 75% by weight of the total
protein, with at least about 80% being preferred, and at least
about 90% being particularly preferred. Alternatively, the target
protein need not be substantially pure as long as the sample
comprising the target protein is substantially free of other
components that can contribute to the production of ADP or
phosphate (or, in the case of indirect assays, other components
which are subsequently assayed).
The target proteins may be isolated or purified in a variety of
ways known to those skilled in the art depending on what other
components are present in the sample. Standard purification methods
include electrophoretic, molecular, immunological and
chromatographic techniques, including ion exchange, hydrophobic,
affinity, and reverse-phase HPLC chromatography, and
chromatofocusing. For example, the target protein may be purified
using a standard anti-target antibody column. Ultrafiltration and
diafiltration techniques, in conjunction with protein
concentration, are also useful. For general guidance in suitable
purification techniques, see Scopes, R., Protein Purification,
Springer-Verlag, N.Y. (1982).
Suitable purification schemes for some specific kinesins are
outlined in U.S. Ser. No. 09/295,612, filed Apr. 20, 1999, hereby
expressly incorporated herein in its entirety, along with
referenced materials.
The present invention provides methods for screening for modulators
of target proteins. By "modulators" herein is meant both
antagonists and agonists of the target protein. Thus, "modulating
the activity of the target protein" includes an increase in target
protein activity, a decrease in target protein activity, or a
change in the type or kind of activity present. Generally, the
modulator will both bind to the target protein (although this may
not be necessary), and alter its biological or biochemical activity
as defined herein. For inhibitors, changes of 25%, 50%, 75% and
most preferably 100% of at least one biological activity of the
target protein is seen. For activators, preferably the change is a
change of at least 40%, more preferably at least 60%, more
preferably at least 80%, more preferably at least 100%, more
preferably at least 200%, and most preferably by at least 500%.
Accordingly, the present invention provides methods for screening
candidate bioactive agents for the ability to modulate a target
protein's activity. By "candidate agent" or "candidate bioactive
agent" or "drug candidate" or grammatical equivalents herein is
meant any molecule, e.g., protein, oligopeptide, small organic
molecule, polysaccharide, polynucleotide to be tested in a
screening assay.
Candidate agents encompass numerous chemical classes, though
typically they are organic molecules, preferably small organic
compounds having a molecular weight of more than 100 and less than
about 2,500 daltons. Candidate agents comprise functional groups
necessary for structural interaction with proteins, particularly
hydrogen bonding, and typically include at least an amine,
carbonyl, hydroxyl or carboxyl group, preferably at least two of
the functional chemical groups. The candidate agents often comprise
cyclical carbon or heterocyclic structures and/or aromatic or
polyaromatic structures substituted with one or more of the above
functional groups. Candidate agents are also found among
biomolecules including peptides, saccharides, fatty acids,
steroids, purines, pyrimidines (including derivatives, structural
analogs, or combinations thereof), derivatives, structural analogs
or combinations thereof.
Candidate agents are obtained from a wide variety of sources
including libraries of synthetic or natural compounds. For example,
numerous means are available for random and directed synthesis of a
wide variety of organic compounds and biomolecules, including
expression of randomized oligonucleotides. Alternatively, libraries
of natural compounds in the form of bacterial, fungal, plant and
animal extracts are available or readily produced. Additionally,
natural or synthetically produced libraries and compounds are
readily modified through conventional chemical, physical and
biochemical means. Known pharmacological agents may be subjected to
directed or random chemical modifications, such as acylation,
alkylation, esterification, amidification to produce structural
analogs.
In an embodiment provided herein, the candidate bioactive agents
are proteins. The protein may be made up of naturally occurring
amino acids and peptide bonds, or synthetic peptidomimetic
structures. For example, homo-phenylalanine, citrulline and
noreleucine are considered amino acids for the purposes of the
invention. "Amino acid" also includes imino acid residues such as
proline and hydroxyproline. The side chains may be in either the
(R) or the (S) configuration. In the preferred embodiment, the
amino acids are in the (S) or L-configuration. If non-naturally
occurring side chains are used, non-amino acid substituents may be
used, for example to prevent or retard in vivo degradations.
In another embodiment, the candidate bioactive agents are naturally
occurring proteins or fragments of naturally occurring proteins.
Thus, for example, cellular extracts containing proteins, or random
or directed digests of proteinaceous cellular extracts, may be
used. In one embodiment, the libraries are of bacterial, fungal,
viral, and mammalian proteins, with the latter being preferred, and
human proteins being especially preferred.
In one embodiment, the candidate agents are peptides of from about
2 to about 30 amino acids, with from about 5 to about 20 amino
acids being preferred, and from about 7 to about 15 being
particularly preferred. The peptides may be digests of naturally
occurring proteins as is outlined above, random peptides, or random
peptides. By randomized or grammatical equivalents herein is meant
that each nucleic acid and peptide consists of essentially random
nucleotides and amino acids, respectively. Since generally these
random peptides (or nucleic acids, discussed below) are chemically
synthesized, they may incorporate any nucleotide or amino acid at
any position. The synthetic process can be designed to generate
randomized proteins or nucleic acids, to allow the formation of all
or most of the possible combinations over the length of the
sequence, thus forming a: library of randomized candidate bioactive
proteinaceous agents.
In one embodiment, the library is fully randomized, with no
sequence preferences or constants at any position. In a preferred
embodiment, the library is biased. That is, some positions within
the sequence are either held constant, or are selected from a
limited number of possibilities. For example, in a preferred
embodiment, the nucleotides or amino acid residues are randomized
within a defined class, for example, of hydrophobic amino acids,
hydrophilic residues, sterically biased (either small or large)
residues, towards the creation of cysteines, for cross-linking,
prolines for SH-3 domains, serines, threonines, tyrosines or
histidines for phosphorylation sites, etc., or to purines, etc.
In another embodiment, the candidate agents are nucleic acids. By
nucleic acid or "otigonucleotide" or grammatical equivalents herein
means at least two nucleotides covalently linked together. A
nucleic acid of the present invention will generally contain
phosphodiester bonds, although in some cases, as outlined below,
nucleic acid analogs are included that may have alternate
backbones, comprising, for example, phosphoramide (Beaucage et at.,
Tetrahedron 49(10):1925 (1993) and references therein; Letsinger,
J. Org. Chem. 35:3800 (1970); Sprinzl et al., Eur. J. Biochem.
81:579 (1977); Letsinger et al., Nucl. Acids Res. 14:3487 (1986);
Sawai et al, Chem. Lett. 805 (1984), Letsinger et al., J. Am. Chem.
Soc. 110:4470 (1988); and Pauwels et al., Chemica Scripta 26:141
91986)), phosphorothioate (Mag et al., Nucleic Acids Res. 19:1437
(1991); and U.S. Pat. No. 5,644,048), phosphorodithioate (Briu et
al., J. Am. Chem. Soc. 11 1:2321 (1989), O-methylphophoroamidite
linkages (see Eckstein, Oligonucleotides and Analogues: A Practical
Approach, Oxford University Press), and peptide nucleic acid
backbones and linkages (see Egholm, J. Am. Chem. Soc. 114:1895
(1992); Meier et al., Chem. Int. Ed. Engl. 31:1008 (1992); Nielsen,
Nature, 365:566 (1993); Carlsson et al., Nature 380:207 (1996), all
of which are incorporated by reference). Other analog nucleic acids
include those with positive backbones (Denpcy et al., Proc. Natl.
Acad. Sci. USA 92:6097 (1995); non-ionic backbones (U.S. Pat. Nos.
5,386,023, 5,637,684, 5,602,240, 5,216,141 and 4,469,863;
Kiedrowshi et al., Angew. Chem. Intl. Ed. English 30:423 (1991);
Letsinger et al., J. Am. Chem. Soc. 110:4470 (1988); Letsinger et
al., Nucleoside & Nucleotide 13:1597 (1994); Chapters 2 and 3,
ASC Symposium Series 580, Carbohydrate Modifications in Antisense
Research, Ed. Y. S. Sanghui and P. Dan Cook; Mesmaeker et al.,
Bioorganic & Medicinal Chem. Lett. 4:395 (1994); Jeffs et al.,
J. Biomolecular NMR 34:17 (1994); Tetrahedron Lett. 37:743 (1996))
and non-ribose backbones, including those described in U.S. Pat.
Nos. 5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium
Series 580, Carbohydrate Modifications in Antisense Research, Ed.
Y. S. Sanghui and P. Dan Cook. Nucleic acids containing one or more
carbocyclic sugars are also included within the definition of
nucleic acids (see Jenkins et al., Chem. Soc. Rev. (1995)
pp169-176). Several nucleic acid analogs are described in Rawls, C
& E News Jun. 2, 1997 page 35. All of these references are
hereby expressly incorporated by reference. These modifications of
the ribose-phosphate backbone may be done to facilitate the
addition of additional moieties such as labels, or to increase the
stability and half-life of such molecules in physiological
environments.
In addition, mixtures of naturally occurring nucleic acids and
analogs can be made. Alternatively, mixtures of different nucleic
acid analogs, and mixtures of naturally occurring nucleic acids and
analogs may be made. The nucleic acids may be single stranded or
double stranded, as specified, or contain portions of both double
stranded or single stranded sequence. The nucleic acid may be DNA,
both genomic and cDNA, RNA or a hybrid, where the nucleic acid
contains any combination of deoxyribo- and ribo-nucleotides, and
any combination of bases, including uracil, adenine, thymine,
cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine,
isoguanine, etc.
As described above generally for proteins, nucleic acid candidate
agents may be naturally occurring nucleic acids, random nucleic
acids, or biased random nucleic acids. For example, digests of
procaryotic or eukaryotic genomes may be used as is outlined above
for proteins.
In a preferred embodiment, the candidate bioactive agents are
organic chemical moieties, a wide variety of which are available in
the literature.
In a preferred embodiment, the candidate agent is a small molecule.
The small molecule is preferably 4 kilodaltons (kd) or less. In
another embodiment, the compound is less than 3 kd, 2 kd or 1 kd.
In another embodiment the compound is less than 800 daltons (D),
500 D, 300 D or 200 D. Alternatively, the small molecule is about
75 D to 100 D, or alternatively, 100 D to about 200 D.
Devices for the preparation of combinatorial libraries are
commercially available (see, e.g., 357 MPS, 390 NWS, Advanced Chem
Tech, Louisville Ky.; Symphony, Rainin, Woburn, Mass.; 433A Applied
Biosystems, Foster City, Calif.; 9050 Plus, Millipore, Bedford,
Mass.).
The present invention provides methods of screening candidate
bioactive agents for modulators of target protein activity. In a
preferred embodiment, the methods are in vitro methods, utilizing
purified or partially purified target proteins. Alternatively, the
methods are in vivo methods, utilizing cells comprising target
nucleic acids that can be expressed to produce target proteins,
particularly when the target protein is either secreted or on the
surface.
In a preferred embodiment, the methods comprise combining a target
protein and a candidate bioactive agent, and evaluating the effect
on the target protein's activity. By "target protein activity" or
grammatical equivalents herein is meant the biological activity of
the target protein. As will be appreciated by those in the art, the
activity of the target protein will vary with the target protein
chosen, and will be generally ascertainable by one of skill in the
art of the target protein.
In a preferred embodiment, the methods of the invention comprise
the addition of candidate agents to the target proteins. In
general, this is done under conditions which normally allow the
direct or indirect production of ADP or phosphate by the target
protein. The phrase "under conditions which normally allow
production or utilization of ADP or phosphate" as used herein means
that all of the compositions and conditions are provided to allow
the production or utilization of ADP or phosphate. Thus, the
reaction which directly or indirectly produces or uses ADP or
phosphate would normally occur in the absence of the modulator.
As will be appreciated by those in the art, the components are
added in buffers and reagents to assay target protein activity and
give optimal signals (i.e. the largest ADP or phosphate signals
possible). Since the methods outlined herein allow kinetic
measurements, the incubation periods are optimized to give adequate
detection signals over the background.
A "modulator of a target protein which directly or indirectly
produces or uses ADP or phosphate" can be any compound as described
herein in the context of candidate agents which modulates the
target protein's direct or indirect production or use of ADP or
phosphate relative to a control.
In one aspect, the method comprises subjecting the mixture to an
enzymatic reaction which uses ADP or phosphate as a substrate under
conditions which normally allow the ADP or phosphate to be utilized
and determining the level of activity of the enzymatic reaction.
This step can be performed in conjunction with identifying a
modulator of a target protein which directly or indirectly produces
ADP or phosphate or independently thereof to identify a modulator
of a protein which uses ADP or phosphate.
The phrase to "use ADP or phosphate" as used herein means that the
AD? or phosphate are directly acted upon. In one case, the ADP, for
example, can be hydrolyzed or can be phosphorylated. As another
example, the phosphate can be added to another compound. As used
herein, in each of these cases, ADP or phosphate is acting as a
substrate.
There are a number of enzymatic reactions known in the art which
use ADP as a substrate. For example, kinase reactions such as
pyruvate kinases are well known. Nature, 78:632 (1956); Mol.
Pharmacol, 6(1):31-40 (1970). This is a preferred method in that it
allows the regeneration of ATP. In one embodiment, the level of
activity of the enzymatic reaction is determined directly. For
example, in a pyruvate kinase reaction, pyruvate or ATP can be
measured by conventional methods known in the art.
In a preferred embodiment, the level of activity of the enzymatic
reaction which uses ADP as a substrate is measured indirectly by
being coupled to another reaction. For example, in one embodiment,
the method further comprises a lactate dehydrogenase reaction under
conditions which normally allow the oxidation of NADH, wherein said
lactate dehydrogenase reaction is dependent on the pyruvate kinase
reaction. Measurement of enzymatic reactions by coupling is known
in the art, i.e., Nature, 178:632 (1956) and is further discussed
below in regards to fluorescence.
Furthermore, there are a number of reactions which utilize
phosphate. Examples of such reactions include a purine nucleoside
phosphorylase reaction. This reaction can be measured directly or
indirectly. The reaction can be measured directly by conventional
methods known in the art.
In a preferred embodiment, the level of activity of the enzymatic
reaction which uses phosphate as a substrate is measured indirectly
by being coupled to another reaction. For example, in one
embodiment, the method further comprises a purine analog cleavage
reaction under conditions which normally allow the cleavage of the
purine analog. See, PNAS, 89:4884-4887 (1992); Anal. Biochem.,
246:86-95 (1997); Biochem., J., 266:611-614 (1990). Alternatively,
xanthine oxidase can be used in conjunction with purine nucleoside
phosphorylase to couple phosphate production to a change in the
absorbance of a substrate for xanthine oxidase. Clin. Chim. Acta.,
223:149-157 (1993).
In one embodiment, the detection of the ADP or phosphate proceeds
non-enzymatically, for example by binding or reacting the ADP or
phosphate with a detectable compound. For example, phosphomolybdate
based assays may be used which involve conversion of free phosphate
to a phosphomolybdate complex. J. Biol. Chem., 66:375-400 (1925).
One method of quantifying the phosphomolybdate is with malchite
green. Chin. Chim. Acta, 14:361-366 (1966). Alternatively, a
fluorescently labeled form of a phosphate binding protein, such as
the E. coli phosphate binding protein, can be used to measure
phosphate by a shift in its fluorescence.
In a preferred embodiment, detection of the assay is done using a
detectable label. By "labeled" herein is meant that a compound has
at least one element, isotope or chemical compound attached to
enable the detection of the compound. In general, labels fall into
three classes: a) isotopic labels, which may be radioactive or
heavy isotopes; b) magnetic, electrical, thermal; and c) colored or
luminescent dyes; although labels include enzymes and particles
such as magnetic particles as well. The dyes may be chromophores or
phosphors but are preferably fluorescent dyes, which due to their
strong signals provide a good signal-to-noise ratio for detection.
Suitable dyes for use in the invention include, but are not limited
to, fluorescent lanthanide complexes, including those of Europium
and Terbium, fluorescein, rhodamine, tetramethylrhodamine, eosin,
erythrosin, coumarin, methyl-coumarins, pyrene, Malacite green,
stilbene, Lucifer Yellow, Cascade Blue.TM., Texas Red, and
derivatives thereof, and others described in the 6th Edition of the
Molecular Probes Handbook by Richard P. Haugland, hereby expressly
incorporated by reference.
The invention provides methods of screening candidate agents for
the ability to serve as modulators of target protein activity. In a
preferred embodiment, high throughput screening (HTS) systems are
used, which can include the use of robotic systems. The assays of
the present invention offer the advantage that many samples can be
processed in a short period of time. For example, plates having 96
or as many wells as are commercially available can be used.
High throughput screening systems are commercially available (see,
e.g., Zymark Corp., Hopkinton, Mass.; Air Technical Industries,
Mentor, Ohio; Beckman Instruments, Inc., Fullerton, Calif.;
Precision Systems, Inc., Natick, Mass., etc.) These systems
typically automate entire procedures including all sample and
reagent pipetting, liquid dispensing, timed incubations, and final
readings of the microplate in detector(s) appropriate for the
assay. These configurable systems provide high throughput and rapid
start up as well as a high degree of flexibility and customization.
The manufacturers of such systems, i.e., Zymark Corp., provide
detailed protocols for the various high throughput assays.
Generally a plurality of assay mixtures are run in parallel with
different agent concentrations to obtain a differential response to
the various concentrations. Typically, one of these concentrations
serves as a negative control, i.e., at zero concentration or below
the level of detection. However, in one embodiment, any
concentration can be used as the control for comparative
purposes.
In one preferred embodiment, high throughput screening methods
involve providing a library containing a large number of compounds
(candidate compounds) potentially having the desired activity. Such
"combinatorial chemical libraries" are then screened in one or more
assays, as described herein, to identify those library members
(particular chemical species or subclasses) that display a desired
characteristic activity. The compounds thus identified can serve as
conventional "lead compounds" or can themselves be used as
potential or actual therapeutics or agricultural compounds.
For example, in one embodiment, candidate agents are assayed in
highly parallel fashion by using multiwell plates by placing the
candidate agents either individually in wells or testing them in
mixtures. Assay components, such as for example, molecular motors,
protein filaments, coupling enzymes and substrates, and ATP can
then be added to the wells and the absorbance or fluorescence of
each well of the plate can be measured by a plate reader. A
candidate agent which modulates the function of the molecular motor
is identified by an increase or decrease in the rate of ATP
hydroylsis compared to a control assay in the absence of that
candidate agent.
A preferred HTS system is as follows. The system comprises a
microplate input function which has a storage capacity matching a
logical "batch" size determined by reagent consumption rates. The
input device stores and, delivers on command, barcoded assay plates
containing pre-dispensed samples, to a barcode reader positioned
for convenient and rapid recording of the identifying barcode. The
plates are stored in a sequential nested stack for maximizing
storage density and capacity. The input device can be adjusted by
computer control for varying plate dimensions. Following plate
barcode reading, the input device can be adjusted by computer
control for varying plate dimensions. Following plate barcode
reading, the input device transports the plate into the pipetting
device which contains the necessary reagents for the assay.
Reagents are delivered to the assay plate with the pipetting
device. Tip washing in between different reagents is performed to
prevent carryover. A time dependent mixing procedure is performed
after each reagent to effect a homogeneous solution of sample and
reagents. The sequential addition of the reagents is delayed by an
appropriate time to maximize reaction kinetics and readout levels.
Immediately following the last reagent addition, a robotic
manipulator transfers the assay plate into an optical interrogation
device which records one or a series of measurements to yield a
result which can be correlated to an activity associated with the
assay. The timing of the robotic transfer is optimized by
minimizing the delay between "last reagent" delivery and transfer
to the optical interrogation device. Following the optical
interrogation, the robotic manipulator removes the finished assay
plates to a waste area and proceeds to transfer the next plate from
pipetting device to optical interrogation device. Overlapping
procedures of the input device, pipetting device and optical
interrogation device are used to maximize throughput.
It is understood that the methods provided herein can be applied to
a varied array of target proteins and are not limited to
cytoskeletal component systems. However, for illustrative purposes,
another example of the present invention is to assay for modulators
of the polymerized state of cytoskeletal filament proteins actin or
tubulin. In this example, the candidate agent or mixture comprising
at least one candidate agent is incubated with the filament protein
under conditions that would normally promote either polymerization
or depolymerization. A molecular motor that is activated by the
filament is then added to the assay mixture and its activity is
monitored by ADP or phosphate release as discussed above. Candidate
agents which increase the fraction of the filament protein in a
polymerized state will be identified by an increase in the motor
ATPase and those which increase the fraction of the filament
protein in a depolymerized state will be identified by a decrease
in the motor ATPase.
It is understood that once a modulator or binding agent is
identified that it can be subjected to further assays to further
confirm its activity. In particular, the identified agents can be
entered into a computer system as lead compounds and compared to
others which may have the same activity. The agents may also be
subjected to in vitro and preferably in vivo assays to confirm
their use in medicine as a therapeutic or diagnostic or in the
agricultural arena.
In a preferred embodiment, approximately 1000 assays are performed
per hour with very low false negative and false positive rates,
with up to 10,000 assays an hour being preferred and more than
10,000 assays per hour being particularly preferred. In a
particularly preferred embodiment, at least one or more of the
steps regarding automated liquid handling or preferred assay design
as described herein are included.
In one embodiment, the method comprises automated liquid
handling.
In preferred embodiment, an antifoam or a surfactant is included in
the assay mixture and wash solution. Suitable antifoams include,
but are not limited to, antifoam 289 (Sigma), and others
commercially available. Suitable surfactants include, but are not
limited to, Tween, Tritons including Triton X-100, saponins, and
polyoxyethylene ethers. This eliminates bubbles which often result
in conventional methods requiring pipetting into low volume assay
wells. Thus, in a preferred embodiment, the invention includes the
use of an antifoam, detergent or surfactant as a reagent in a high
throughput screens, including, but not limited to the screens of
the invention. Generally the antifoams, detergents or surfactants
are added at a range from about 0.01 ppm to about 10 ppm, with from
about 1 to about 2 ppm being preferred. In a further preferred
embodiment, the invention includes the use of an antifoam,
surfactant or detergent when the assay requires mixing,
particularly physical mixing such as shaking the microtiter plates.
In an additional preferred embodiment, the invention includes the
use of an antifoam, surfactant or detergent when the assay is done
in microtiter plates, particularly plates with 96 wells or more,
including 96, 384 and 1536 plates.
In another aspect, a round sample well is used. This helps increase
the pathlength for absorbance measurements for a given assay volume
and helps flatten the meniscus of the solution in each assay well.
Preferably, the method comprises vigorous shaking of the sample
plate following the addition of each reagent.
In a preferred embodiment herein, a preferred assay design is
provided. In one aspect, the preferred assay preferably uses a
multi-time-point (kinetic) assay, with at least two data points
being preferred. As will be appreciated by those in the art, the
interval can be adjusted to correlate with the biological activity
of the protein. In the case of multiple measurements the absolute
rate of the protein activity can be determined, and such
measurements have higher specificity particularly in the presence
of candidate agents which have similar absorbance or fluorescence
properties to that of the enzymatic readout. The kinetic assay
reduces the false positive rate. In an additional aspect, the
kinetic rate are normalized to several control wells on each assay
plate. This allows for some variation in the activity of the target
proteins and the stability of assay reagents over time and thus
permits screening runs of several hours.
When proteins that use ATP are included, the pyruvate
kinase/lactate dehydrogenase embodiments are particularly preferred
due to the advantage of ATP regeneration so that ATP concentration
is constant over time.
Further regarding variation of the assays, it is understood that
for a kinesin-microtubule modulator assay, the order of addition of
the assay components affects the ATPase rate.
The invention further provides methods for identifing whether any
two test proteins interact. Briefly, the assay is functionally
similar to a yeast two-hybrid system, but relies on an increase in
ATPase activity as a result of bringing two components together as
a result of a protein-protein interaction. As an example, the
system is described using a biological polymer binding site and a
polymer stimulated ATPase, although as will be appreciated by those
in the art, any two components that result in an increase in ATPase
activity as a result of association can be used. For example, a
first test protein (a "bait" protein), for which an interaction is
sought, is joined, usually covalently, to a biological polymer
binding protein, for example a cytoskeletal binding protein (such
as a microtubule binding protein) to form a first target chimera.
The term "chimera" or "fusion protein" as used herein refers to a
protein (polypeptide) composed of two polypeptides that, while
typically unjoined in their native state, typically are joined by
their respective amino and carboxyl termini through a peptide
linkage to form a single continuous polypeptide. It will be
appreciated that the two all polypeptide components can be directly
joined or joined through a peptide linker/spacer.
A second test protein (a "prey" protein), is joined, again usually
covalently, to an ATPase domain that is stimulated by the
cytoskeletal component to form a second target chimera. Upon
combination with the cytoskeletal component, the first target
chimera binds to the cytoskeletal component, and if the first and
second target proteins interact, the second target chimera is
brought into proximity with the cytoskeletal component, and thus
the ATPase activity is stimulated and can be detected. If there is
no interaction, no increase in ATP production is observed.
In a preferred embodiment, the biological polymer binding protein
comprises just a domain of a larger protein that comprises an
ATPase domain; that is, the ATPase domain has been removed.
Alternatively, the biological polymer binding protein may include
the larger protein but have the ATPase domain inactivated, for
example by mutation. Similarly, the ATPase domain may be either
just the ATPase functional domain of a protein, or it may include a
larger protein that has the binding domain inactivated.
As discussed above, the chimera proteins are generally joined
covalently, for example by making fusion proteins, although
covalent cross-linking can be used, or high affinity non-covalent
associations can also be done, for example using binding partners
such as biotin/avidin, etc. In a preferred embodiment, the fusion
proteins are made using fusion genes, as is generally known in the
art.
In a preferred embodiment, the target proteins should not have
ATPase activity themselves, although it is possible to detect
increases in activity.
Suitable biological polymers include, but are not limited to,
nucleic acids including DNA and RNA, and cytoskeletal components
including, but not limited to, microtubules and microfilaments
(actin filaments).
Suitable biological binding sites include, but are not limited to,
nucleic acid binding domains (when nucleic acids are the biological
polymer), and molecular motor binding domains (in the case of
cytoskeletal components).
Suitable ATPases include, but are not limited to, those that
exhibit an increase (stimulation) in the presence of the
biopolymer, such as DNA and RNA polymerases in the case of nucleic
acids, microtubule stimulated ATPases in the case of microtubules
including kinesins and dyneins, and actin stimulated ATPases such
as myosins.
In a preferred embodiment, the first test protein is attached to a
functional molecular motor binding domain to provide a first target
chimera. The second test protein is attached to a functional
microtubule stimulated ATPase domain to form a second target
chimera. The first and second target chimeras are combined under
conditions which normally allow activity of a motor protein which
comprises a molecular motor binding domain and a microtubule
stimulated ATPase domain. An increase in motor protein activity
indicates interaction between the two test proteins.
Customarily one bait protein is used to test a library of test
sequences as is described below; however, as will be appreciated by
those in the art, the bait protein may be one of a library as well,
thus forming an experimental matrix wherein two libraries (although
the coding regions of the libraries could be identical) are
evaluated for protein-protein interactions. In a preferred
embodiment, self-activating bait proteins are filtered out from the
bait protein library.
In another embodiment a method for identifying whether a candidate
agent is a modulator of at least one of a first and second test
protein is provided. In this case, a candidate agent is combined
with the first and second chimeras as described above. A change in
molecular motor activity in the presence and absence of the
candidate agent indicates that the candidate agent is a modulator
of at least one of the two candidate agents.
Thus, the chimeras of the present invention can be formed used
recombinant techniques known in the art. The chimera can be formed
at the protein level wherein two polypeptides are joined, or at the
molecular level wherein a nucleic acid is formed which encodes the
appropriate functional motor component and the appropriate test
protein.
In a preferred embodiment, the nucleic acids encoding a chimera are
used to express the respective recombinant chimera. A variety of
expression vectors, including viral and non-viral expression
vectors can be made which are useful for recombinant protein
expression in a variety of systems, including, but not limited to,
yeast, bacteria, archaebacteria, fungi, insect cells and animal
cells, including mammalian cells.
The expressed chimera may also include farther fusion domains
including tag polypeptides. Recombinant protein is produced by
culturing a host cell transformed with a nucleic acid encoding the
chimera (generally as an expression vector), under the appropriate
conditions that induce or cause expression of the chimera.
In a preferred embodiment, the recombinant chimera is purified
following expression, as outlined above.
For using the chimeras in the assays described herein, if the two
test proteins bind to one another, a complex with both chimeras
comprising a functional molecular motor is formed. Thus, the
binding interaction between the two test proteins can be identified
by functional motor activity under conditions which would normally
allow motor activity if both a functional microtubule stimulated
ATPase and binding domain were present.
In the case of identifying a modulator in an assay utilizing the
chimeras of the present invention, the modulator can be an
activator of the motor activity. Thus, in the absence of the
candidate agent, there may be no motor activity, however, in the
presence of the candidate agent, motor activity occurs. Conversely,
there may be significant motor activity, indicating that the two
testbinding proteins interact, but this may decrease in the
presence of a candidate agent. In either case, the candidate agent
is identified as a modulator of at least one the two test
proteins.
In a preferred embodiment, motor activity is identified by ATP
hydrolysis as described above. However, it is understood that motor
activity can be identified by a number of assays. Such assays
include microtubule gliding, depolymerization/polymerization and
any motor activity which requires both binding and ATPase activity.
Therefore, in the case that the molecular motor used has another
specific activity, such as involvement in mitosis or axonal
transport, specific assays for those activities can be
utilized.
Generally motility assays involve immobilizing one component of the
system (e.g., the kinesin motor or the microtubule) and then
detecting movement, or change thereof, of the other component.
Thus, for example, in a preferred embodiment, the microtubule will
be immobilized (e.g., attached to a solid substrate) and the
movement of the kinesin motor molecule(s) will be visually
detected. Typically the molecule that is to be detected is labeled
(e.g., with a fluorescent label) to facilitate detection.
Methods of performing motility assays are well known to those of
skill in the art (see, e.g., Hall, et al. (1996), Biophys. J., 71:
3467-3476, Turner et al., 1996, Anal. Biochem. 242 (1):20-5; Gittes
et al., 1996, Biophys. J. 70(1): 418-29; Shirakawa et al., 1995, J.
Exp. Biol. 198: 1809-15; Winkelmann et al., 1995, Biophys. J. 68:
2444-53; Winkelmann et al., 1995, Biophys. J. 68: 72S, and the
like).
In addition to the assays described above for identifying ATPase
activity, conventional methods can be used. For example, P.sub.i
release from kinesin can be quantified. In one preferred
embodiment, the ATPase activity assay utilizes 0.3 M PCA
(perchloric acid) and malachite green reagent (8.27 mM sodium
molybdate II, 0.33 mM malachite green oxalate, and 0.8 mM Triton
X-100). To perform the assay, 10 .mu.L of reaction is quenched in
90 .mu.L of cold 0.3 M PCA. Phosphate standards are used so data
can be converted to mM inorganic phosphate released. When all
reactions and standards have been quenched in PCA, 100 .mu.L of
malachite green reagent is added to the to relevant wells in e.g.,
a microtiter plate. The mixture is developed for 10-15 minutes and
the plate is read at an absorbance of 650 nm. If phosphate
standards were used, absorbance readings can be converted to mM
P.sub.i and plotted over time.
Additionally, in the case of methods provided herein utilizing the
chimeras in accordance with the present invention, the remaining
ATP can be measured using the luciferin-luciferase system. Anal.
Biochem., 40:1-17 (1971).
The assays are preferably performed in a high throughput system as
described herein utilizing multiwell plates and fluorescence or
absorbance readouts.
It is understood that the examples and embodiments described herein
are for illustrative purposes only and that various modifications
or changes in light thereof will be suggested to persons skilled in
the art and are to be included within the spirit and purview of
this application and scope of the appended claims. All
publications, patents, and patent applications cited herein are
hereby incorporated by reference in their entirety.
EXAMPLE
A High Throughput Assay for Modulators of the Molecular Motor
Kinesin
This assay is based on detection of ADP production from kinesin's
microtubule stimulated ATPase. ADP production is monitored by a
coupled enzyme system consisting of pyruvate kinase and lactate
dehydrogenase. Under the assay conditions described below, pyruvate
kinase catalyzes the conversion of ADP and phosphoenol pyruvate to
pyruvate and ATP. Lactate dehydrogenase then catalyzes the
oxidation-reduction reaction of pyruvate and NADH to lactate and
NAD+. Thus, for each molecule of ADP produced, one molecule of NADH
is consumed. The amount of NADH in the assay solution is monitored
by measuring light absorbance at a wavelength of 340 nm.
Assay components
A kinesin heavy chain construct consisting of the N-terminal 420
amino acids is used in the assay. The final 25 .mu.l assay solution
consists of the following: 5 .mu.g/ml kinesin, 30 .mu.g/ml
microrubules, 5 .mu.M Taxol, 0.8 mM NADH, 1.5 mM phosphoenol
pyruvate, 3.5 U/ml pyruvate kinase, 5 U/ml lactate dehydrogenase,
25 mM Pipes/KOH pH 6.8, 2mM MgCl2, 1 mM EGTA, 1 m MDTT, 0.1 mg/ml
BSA, 0.001% antifoam 289 (Sigma), and 1 mM ATP.
Compound plates
Potential chemical modulators of kinesin are dissolved in DMSO at a
concentration of approximately 1 mg/ml, and 0.5 .mu.l of each
chemical solution is dispensed into a single well of a clear 384
well plate (Clinipate, Labsystems) On each plate, there are at
least 16 wells into which pure DMSO (without a candidate compound)
is dispensed. These wells serve as negative controls for comparison
to the potential chemical modulators on that plate. The compound
plates are made in advance and stored at 4.degree. C., and each
plate is labeled with a bar code which is used to identify the
compounds on a given plate.
Instrumentation
The robotic system that runs the assay consists of a plate storage
and retrieval device (Plate Stak, CCS Packard), a 96 channel
automated pipetting device (Multimek, Beckman), a robotic arm
(Twister, Zymark), and a plate reader for absorbance (Ultramark,
BioRad). The system is controlled by a custom-built software
application.
Assay Performance
A stack of compound plates is placed in the plate storage devices
and plates are transferred one at a time to the automated pipetting
device by the plate carrier of the Plat Stak. Each of the 384 wells
are then filled with 20 .mu.l of a solution consisting of all of
the assay components described above except for ATP. The plate is
then agitated at high frequency by rapidly moving the plate carrier
between two positions that are separated by a few millimeters. The
plate is then returned to the pipetting position. While the shaking
of the plate occurs, the pipet tips are washed with a solution of
0.001% antifoam in deionized water. To start the assay, 5 .mu.l of
a second solution containing ATP is then added to each well. The
solution is then mixed by a second cycle of high frequency
agitation. The plate is then transferred to the plate reader by the
robotic arm. In the plate reader, 10 absorbance measurements at 340
nm are taken at 12 second intervals to produce a 2 minute kinetic
read for each well. While one plate is being read, the next plate
is transferred to the pipetting device and prepared up to but not
including the addition of the second solution. When the plate read
is complete, the robotic arm transfers the plate to a waste chute
and simultaneously the second solution is pipetted into the next
plate so that it can be transferred to the reader to complete the
cycle. The entire assay is run at room temperature
.about.20.degree. C.
Data analysis
Following data acquisition, the maximum rate of the absorbance
change is calculated for each well and normalized to the average of
the control wells (without compound) which were present on the same
plate. The normalized rates are then entered into an Oracle
database, and this allows them to be correlated with the potential
chemical modulators. On each plate, the coefficient of variation of
the slopes for the control wells ranges from 4-8%. Quality control
is assured by monitoring for a minimal initial absorbance and a
linear absorbance change.
Important features
There are several features of this system which are important. The
kinetic design which consists of multiple absorbance measurements
dramatically improves the specificity of the assay over a single
endpoint measurement. First, the rate of the reaction is to a first
approximation independent of small differences between wells in the
time from the start of the reaction to the first reading, and as a
result, the overall variation in the data is reduced. Second, the
rate of the absorbance change is not affected by having a chemical
compound which absorbs light of the same wavelength.
The presence of control wells in each plate and the subsequent
normalization of the data to those wells allows data to be taken
for several hours despite some degradation of the enzyme activities
which results from the aging of the solutions. This also improves
the reproducibility of the data.
The presence of antifoam in the solution and the tip washing
solution improves overall liquid handling by reducing the number of
trapped bubbles in the small wells and helps flatten the fluid
meniscus in each well for more reliable absorbance measurements.
Additional features which improve liquid handling are the vigorous
shaking of the plate described above; and the round shape of the
wells in the microplates used.
The assay components and the performance of the assay are optimized
together to match the overall read time with the rate of kinesin's
ADP production. In this example, the rate of absorbance change is
approximately 150-250 mOD/min. This corresponds to the production
of approximately 2 .mu.M ADP/sec. In addition to optimizing the
rate of ADP production, the read time must be long enough for the
rate of NADH consumption to reach steady state beyond an initial
lag time of several seconds. In some cases, the order of addition
of the reagents can have a significant affect on the rate of ADP
production. In the above example, the optimal rate is achieved by
premixing all reagents except for the compound of interest and
ATP.
* * * * *